We previously demonstrated that cell-cell communication mediated by gap junctions composed of connexin43 was intricately regulated by cAMP- dependent protein kinase and protein kinase C. This regulation appeared to occur by phosphorylation of serine residues in the 362-374 carboxyl tail domain. In a recently completed study, it was found that 6 heart transplant patients diagnosed with visceroatrial heterotaxia (VAH) had mutations in their connexin43 gene, which codes for the predominant gap junction protein of the heart. Five of the six patients had the same mutation: a substitution of Serine 364 by Proline. Twenty-three additional transplant patients without VAH and twenty-five normal controls had no mutations of connexin43. To evaluate whether the mutations had a physiologic impact, site-directed mutagenesis was used to construct expression plasmids for normal connexin43 or each mutation found in VAH patients. Cells that have low levels of endogenous connexin43 but make very few gap junctions and communicate poorly were transfected with normal or Ser364 yields Pro mutant connexin43. Transfectants produced connexin43 at 20 times the level of non- transfected cells and engaged in cell-cell communication to a significantly greater degree that non-transfected cells. However, in cells expressing Ser364 yields Pro mutant connexin43, the microinjection of purified cAMP-dependent protein kinase had no effect, although it significantly increased communication in cells with normal connexin43. Injection of protein kinase C had no effect in cells expressing normal connexin43 but significantly enhanced communication in cells with mutant connexin43. These results demonstrate that the Ser364 yields Pro mutation causes cell-cell communication to be inappropriately regulated relative to cells with normal connexin43 gap junctions, suggesting that the mutations found in VAH patients may, in fact, cause the severe heart malformations. To test this further, we propose to determine whether or not the other connexin43 mutations found in VAH patients may also affect the regulation of cell-cell communication. To do so, cells transfected with normal or mutant connexin43 DNA will be used for microinjection, to analyze the regulation of communication in living cells, and for in vitro and in vivo phosphorylation. These observations are the first evidence that mutations in a gap junction gene coincide with heart malformations in humans. More importantly, they reveal a probable mechanistic basis for these defects, misregulation of cell-cell communication, As such, the results should prove useful to understanding the mechanisms that underlie heart malformation and dysfunction.